Eighty years ago, Arlindo de Assis, an assistant
of the Instituto Vital Brasil published a long article entitled "O immunodiagnostico
da syphilis na actualidade" (The current immune diagnosis of syphilis),
in which he reported and discussed about several serological techniques that
were under development in the world in the distant year of 1925. Since it is
not our purpose to summarize the full text, we cite some parts of the article
showing the author's concerns.1

..."The experience progressively taught us
that the serodiagnosis of syphilis is based on abnormal reactions of organic
liquids which are modified by the disease, over colloidal suspensions of certain
lipids. There are strong reasons to suspect that such reactions have specific
nature; the unexpected aspect of the phenomenon and the unique complexity
of its individualization created, however, in the beginning, severe and compromising
misunderstandings; however, the continued improvement of the technique and
of interpretations have corrected them and gradually headed towards their
elimination."

..."Actually, the contact of the colloidal
system present in the fluids of healthy individuals or, in general, not suffering
from syphilis, is not accompanied by any significant consequence; the colloidal
mixture of these lipids with normal liquids indistinctly continues to flocculate
at a slow pace, like any other similar systems.

Syphilis, nevertheless, changes the interior
medium in such a manner that, from then on, the contact of fluids with lipids
triggers a huge unbalance between the two existing colloidal systems, thus
making flocculation significantly faster."

..."In general practice, the [methods] most
often used are the extracts of ox heart, particularly when carefully added
with cholesterin, as taught by Sachs, in 1917. These are followed by human
heart, syphilitic liver (the so-called abundance of treponemas in the organs
seems to be not important at all), and guinea pig heart."

..."However, we do not intend to deny that
Wassermann's reaction, or its variants, may be positive in the absence of
syphilis and in other diseases. But these conditions are much less frequent
than what is stated, and their diagnosis is relatively easy [to make]: treponemotasis
(yaws) and some cases of tuberous leprosy."

..."In other infectious diseases (paludism,
tuberculosis, gonorrhea, pneumococcal infections), in some constitutional
affections (diabetes), in transient intoxication states (alcoholism, narcosis)
and even in certain physiologic conditions (pregnancy, digestive period),
some false reactions have been observed."

..."III - Flocculation reactions - The
flocculation processes could not replace Wassermann's reaction yet. However,
there were many cases of syphilis with a negative reaction and positive flocculation
processes. Therefore, it is recommended to associate both reactions."

..."Hence, everything tends to prove that,
if the mechanism of fluid changes in syphilis made general and speculative
progresses, in practice, the most loyal demonstration of such changes is the
hemolysis test, discovered by Wassermann. These tests would be even better
if they could appropriately combine with a uniform technique. Flocculation
and turbidity should become practice, since it is the path to improvement;
they correspond to facts of safe observation and probably correct interpretation.
However, at this moment, it would be more prudent to consider Wassermann's
reaction as the basis for the immune diagnosis of lues."

Eighty years later, the serological reactions
with lipid antigens are still useful and important for diagnosis and control
of cure. Throughout those years, the treponemic tests were added to the diagnostic
armamentarium of syphilis, together with, in the past decade, the nucleotide
amplification methods.

LIPID OR REAGINIC TESTS

These tests detect IgG and IgM antibodies against
serum lipids due to damage to the mitochondrial membrane of the host, and against
lipids in the membrane of Treponema pallidum. The flocculation reactions
- particularly VDRL - replaced complement fixation reactions, which were first
described by Wassermann, Neisser & Bruck, in 1906. Even among several other
flocculation reactions reported (Hinton, Kline, Khan, Mazzini, Meinicke), the
VDRL remains predominant,2 and is recommended by the World Health
Organization. Its antigen is uniform in all laboratories since 1941, when Pangborn
isolated the active ingredients of the extract of ox heart used at that time.
The major setback is the need to prepare the mixture of cardiolipin (0.03%),
cholesterol (0.9%) and lecitin (0.21%) every day.

The VDRL test becomes positive five to six weeks
after infection; therefore, it may be negative in case of short-duration chancre.
It is highly sensitive**
in secondary syphilis (100%), and sensibility drops to 70% in late forms. The
rare false-negative cases result from excess antibodies that induce a technical
failure known as prozone phenomenon, which is more marked in cases of syphilis
associated with Aids.

It is highly specific (99-100%) in the healthy
population, and there are false-positive reactions in several morbid conditions
(Chart 1).

It is useful to control cure and the serological
follow-up after treatment should be performed every three months in the first
year, and every month in the second year, when the test will be negative or
there will be persistent low titers (serological scar).3 Serological
scar is considered the persistence of reagins in low titers (of pure serum up
to 1:4) after two years, with positive treponemic tests. Persistent high titer
serology, even with normal CSF, should be followed up longer due to possible
existence of other treponema reservoirs. In asymptomatic cases, retreating is
not effective.

The fast macroscopic reading tests play an important
role in population detection and in clinics for asymptomatic homosexual and
bisexual individua.4 The rapid plasma reagin test (RPR) uses charcoal
particles as an indicator; the screening test (RST) uses Sudan Black B, a liposoluble
dye; and the toluidine red unheated serum test (TRUST) uses an azo pigment.
The future trend of these fast tests is to be replaced by treponema antigens.

TREPONEMIC TESTS

The major difficulty in a developing a better
use of Treponema pallidum as an antigen is the absence of in vitro
culture. In 1949, Nelson and Meyer developed the immobilization test (TPI),
which became the gold standard to diagnose syphilis. Its expensive and demanding
performance restricted its use for academic purposes. To obtain live treponemas,
it is necessary to make several inoculations in rabbit testes (Nichols strains,
1917), which will take from 11 to 28 days to develop orchitis. The live treponemas
obtained are then immobilized by the presence of serum antibodies: if 50% or
more are immobilized, the result is positive5 (disease), if less
than 20%, it is negative. The test is highly sensitive and specific (99%), and
the presence of antibiotics in serum leads to false-negative results. The practical
difficulties led to developing the complement fixation reaction with dead treponemas
(Reiter), which is highly unspecific. The following reactions were specific:
hemagglutination (Rathlev, 1967)6 and FTA-abs. The tests that preceded
the development of FTA-abs reacted to commensal antibodies, and this problem
was initially solved by diluting serum (FTA-200), and later, by absorption with
Reiter's treponema. It presents sensibility of 99.5% and specificity of 88.7%.
In cases of autoimmune diseases it is false negative. Today, the hemagglutination
reactions (MHA-Tp and HA-Tp) are used as confirming tests, mainly if the laboratory
is not equipped with fluorescence microscopy.

The enzyme immunoassay (EIA) uses treponema antigen,
human antiglobulin and enzyme substract, and may be modified to detect IgM.

Western blot technique identifies antibodies
against the following molecular mass immune determinants: 47 KDa, 17 KDa and
15 KDa. Currently, recombinant DNA techniques are used to obtain treponema antigen.7
They are still under investigation, but will be the future commercial option,
since its sensibility is 95.1% and specificity is 94.7%.

The rapid treponemic tests are extremely important
to assist in making diagnosis due to possible immediate reading, thus facilitating
detection. The immune chromatographic assay (Determine® Syphilis
Tp) presents sensibility ranging from 93.7% to 98.4% and specificity, from 95.2%
to 97.3%; it uses a colored treponema-selenium colloidal conjugate as antigen.8,9
Other manufacturer's tests were not very effective, particularly in fingerprick
whole blood investigations.10

Thus, it could be stated that serological diagnosis
is based on two reactions (as mentioned by Assis) - lipid and treponemic - which
are technically more complex. The molecular amplification reactions should be
considered apart.

As early as in 1997, Zoechling et al.11
applied the PCR technique in secondary syphilis lesions (four in six positive)
and in gumma (one in seven). The technique is currently used to detect treponema
antigens in primary syphilis,12 with high sensibility (94.7%) and
specificity (98.6%).13 The RNA amplification is even more sensitive
and reveals the presence of the live microorganism. Chart 2
summarizes the most common approaches to diagnose syphilis in all stages, and
graph 1 demonstrates the behavior of non-treated syphilis.

As we can verify, there has been great progress
in these 80 years; however, in some situations in our daily practice, such developments
seem to not exist. q

Received on April 18, 2005.
Approved by the Editorial Council and accepted for publication on May 09, 2005.

* Work
done at Escola Paulista de Medicina, São Paulo (SP) - Brazil.** By and large, the term sensibility
is used as the capacity to detect real cases of the disease, whereas specificity
means the capacity to not detect healthy people as ill individuals.